Arabidopsis Guard Cells Research Articles

Metabolic modelling reveals distinct roles of sugars and carboxylic acids in stomatal opening and uncovers unexpected carbon fluxes.

Guard cell metabolism is crucial for stomatal dynamics, but a full understanding of its role is hampered by experimental limitations and the flexible nature of the metabolic network. To tackle this challenge, we constructed a time-resolved stoichiometric model of guard cell metabolism that accounts for energy and osmolyte requirements and which is integrated with the mesophyll. The model resolved distinct roles for starch, sugars, and malate in guard cell metabolism and revealed several unexpected flux patterns in central metabolism. During blue light-mediated stomatal opening, starch breakdown was the most efficient way to generate osmolytes with downregulation of glycolysis allowing starch-derived glucose to accumulate as a cytosolic osmolyte. Maltose couldalso accumulate as a cytosolic osmoticum, although this made the metabolic system marginally less efficient. The metabolic energy for stomatal opening was predicted to be derived independently of starch, using nocturnally accumulated citrate which was metabolised in the tricarboxylic acid cycle to malate to provide mitochondrial reducing power for ATP synthesis. In white light-mediated stomatal opening, malate transferred reducing equivalents from guard cell photosynthesis to mitochondria for ATP production. Depending on the capacity for guard cell photosynthesis, glycolysis showed little flux during the day but was crucial for energy metabolism at night. In summary, our analyses have corroborated recent findings in Arabidopsis guard cell research, resolved conflicting observations by highlighting the flexibility of guard cell metabolism, and proposed new metabolic flux modes for further experimental testing.

Endomembrane trafficking driven by microtubule growth regulates stomatal movement in Arabidopsis

Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.

Cytosolic acidification and oxidation are the toxic mechanisms of SO2 in Arabidopsis guard cells.

SO2/H2SO3 can damage plants. However, its toxic mechanism has still been controversial. Two models have been proposed, cytosolic acidification model and cellular oxidation model. Here, we assessed the toxic mechanism of H2SO3 in three cell types of Arabidopsis thaliana, mesophyll cells, guard cells and petal cells. The sensitivity of guard cells of CHLORIDE CHANNEL a (CLCa)-knockout mutants to H2SO3 was significantly lower than those of wildtype plants. Expression of other CLC genes in mesophyll cells and petal cells were different from guard cells. Treatment with antioxidant, disodium 4,5-dihydroxy-1,3-benzenedisulfonate (tiron), increased the median lethal concentration (LC50) of H2SO3 in guard cells indicating the involvement of cellular oxidation, while the effect was negligible in mesophyll cells and petal cells. These results indicate that there are two toxic mechanisms of SO2 to Arabidopsis cells: cytosolic acidification and cellular oxidation, and the toxic mechanism may vary among cell types.

OPEN STOMATA 1 phosphorylates CYCLIC NUCLEOTIDE-GATED CHANNELs to trigger Ca2+ signaling for abscisic acid-induced stomatal closure in Arabidopsis.

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

Signaling pathways involved in microbial indoor air pollutant 3-methyl-1-butanol in the induction of stomatal closure in Arabidopsis.

Indoor air pollution is a global problem and one of the main stress factors that has negative effects on plant and human health. 3-methyl-1-butanol (3MB), an indoor air pollutant, is a microbial volatile organic compound (mVOC) commonly found in damp indoor dwellings. In this study, we reported that 1mg/L of 3MB can elicit a significant reduction in the stomatal aperture ratio in Arabidopsis and tobacco. Our results also showed that 3MB enhances the reactive oxygen species (ROS) production in guard cells of wild-type Arabidopsis after 24h exposure. Further investigation of 24 h 3MB fumigation of rbohD, the1-1, mkk1, mkk3, and nced3 mutants revealed that ROS production, cell wall integrity, MAPK kinases cascade, and phytohormone abscisic acid are all involved in the process of 3MB-induced stomatal. Our findings proposed a mechanism by which 3MB regulates stomatal closure in Arabidopsis. Understanding the mechanisms by which microbial indoor air pollutant induces stomatal closure is critical for modulating the intake of harmful gases from indoor environments into leaves. Investigations into how stomata respond to the indoor mVOC 3MB will shed light on the plant's "self-defense" system responding to indoor air pollution.

Reactive Oxygen Species (ROS) Measurement in Arabidopsis Guard Cells in Response to Biotic and Abiotic Stresses.

One of the major plant stress level indicators is reactive oxygen species (ROS). They have been known to play acentral role in regulating plant responses to various environmental stresses. This book chapter specifically covers abiotic stress induced by a drought hormone abscisic acid and biotic stress induced by Pseudomonas syringe DC3000on single cell-type guard cells. We describe in detail the measurement of ROS production starting from sample preparation to data analysis by fluorescence intensity acquisition using ImageJ software. We discussed the problems faced while performing the experiment and addressed how to overcome them by providing specific guidelines to ensure high quality repeatable data.

Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells.

Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca2+) levels, the interdependence between protons (H+) and Ca2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin HcKCR2 from the pseudofungus Hyphochytrium catenoides, which operates as a H+ conductive, Ca2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of HcKCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca2+ release from the endoplasmic reticulum. A H+-induced cytosolic Ca2+ signal results in membrane depolarization through the activation of Ca2+-dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H+-induced Ca2+ release mechanism in plant cells and establishes HcKCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca2+ signaling.

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, landplants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legumetree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S.saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S.saman.18 We alsodiscovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperaturesensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plantswith temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor.

Stomata are epidermal pores that facilitate plant gas exchange. Grasses have fast stomatal movements, likely due to their dumbbell-shaped guard cells and lateral subsidiary cells. Subsidiary cells reciprocally exchange water and ions with guard cells. However, the relative contribution of subsidiary cells during stomatal closure is unresolved. We compared stomatal gas exchange and stomatal aperture dynamics in wild-type and pan1, pan2, and pan1;pan2 Zea mays (L.) (maize) mutants, which have varying percentages of aberrantly formed subsidiary cells. Stomata with 1 or 2 defective subsidiary cells cannot close properly, indicating that subsidiary cells are essential for stomatal function. Even though the percentage of aberrant stomata is similar in pan1 and pan2, pan2 showed a more severe defect in stomatal closure. In pan1, only stomata with abnormal subsidiary cells fail to close normally. In pan2, all stomata have stomatal closure defects, indicating that PAN2 has an additional role in stomatal closure. Maize Pan2 is orthologous to Arabidopsis GUARD CELL HYDROGEN PEROXIDE-RESISANT1 (GHR1), which is also required for stomatal closure. PAN2 acts downstream of Ca2+ in maize to promote stomatal closure. This is in contrast to GHR1, which acts upstream of Ca2+ , and suggests the pathways could be differently wired.

Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening.

Guard cells control the opening of stomatal pores in the leaf surface, with the use of a network of protein kinases and phosphatases. Loss of function of the CBL-interacting protein kinase 23 (CIPK23) was previously shown to decrease the stomatal conductance, but the molecular mechanisms underlying this response still need to be clarified. CIPK23 was specifically expressed in Arabidopsis guard cells, using an estrogen-inducible system. Stomatal movements were linked to changes in ion channel activity, determined with double-barreled intracellular electrodes in guard cells and with the two-electrode voltage clamp technique in Xenopus oocytes. Expression of the phosphomimetic variant CIPK23T190D enhanced stomatal opening, while the natural CIPK23 and a kinase-inactive CIPK23K60N variant did not affect stomatal movements. Overexpression of CIPK23T190D repressed the activity of S-type anion channels, while their steady-state activity was unchanged by CIPK23 and CIPK23K60N . We suggest that CIPK23 enhances the stomatal conductance at favorable growth conditions, via the regulation of several ion transport proteins in guard cells. The inhibition of SLAC1-type anion channels is an important facet of this response.

COP1 Mediates Dark-Induced Stomatal Closure by Suppressing FT, TSF and SOC1 Expression to Promote NO Accumulation in Arabidopsis Guard Cells

RING-finger-type ubiquitin E3 ligase Constitutively Photomorphogenic 1 (COP1) and floral integrators such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) have been identified as regulators of stomatal movement. However, little is known about their roles and relationship in dark-induced stomatal closure. Here, we demonstrated that COP1 is required for dark-induced stomatal closure using cop1 mutant. The cop1 mutant closed stomata in response to exogenous nitric oxide (NO) but not hydrogen peroxide (H2O2), and H2O2 but not NO accumulated in cop1 in darkness, further indicating that COP1 acts downstream of H2O2 and upstream of NO in dark-induced stomatal closure. Expression of FT, TSF and SOC1 in wild-type (WT) plants decreased significantly with dark duration time, but this process was blocked in cop1. Furthermore, ft, tsf, and soc1 mutants accumulated NO and closed stomata faster than WT plants in response to darkness. Altogether, our results indicate that COP1 transduces H2O2 signaling, promotes NO accumulation in guard cells by suppressing FT, TSF and SOC1 expression, and consequently leads to stomatal closure in darkness. These findings add new insights into the mechanisms of dark-induced stomatal closure.

Multiple cyclic nucleotide-gated channels function as ABA-activated Ca2+ channels required for ABA-induced stomatal closure in Arabidopsis.

Abscisic acid (ABA)-activated inward Ca2+-permeable channels in the plasma membrane (PM) of guard cells are required for the initiation and regulation of ABA-specific cytosolic Ca2+ signaling and stomatal closure in plants. But the identities of the PM Ca2+ channels are still unknown. We hypothesized that the ABA-activated Ca2+ channels consist of multiple CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) proteins from the CNGC family, which is known as a Ca2+-permeable channel family in Arabidopsis (Arabidopsis thaliana). In this research, we observed high expression of multiple CNGC genes in Arabidopsis guard cells, namely CNGC5, CNGC6, CNGC9, and CNGC12. The T-DNA insertional loss-of-function quadruple mutant cngc5-1 cngc6-2 cngc9-1 cngc12-1 (hereafter c5/6/9/12) showed a strong ABA-insensitive phenotype of stomatal closure. Further analysis revealed that ABA-activated Ca2+ channel currents were impaired, and ABA-specific cytosolic Ca2+ oscillation patterns were disrupted in c5/6/9/12 guard cells compared with in wild-type guard cells. All ABA-related phenotypes of the c5/6/9/12 mutant were successfully rescued by the expression of a single gene out of the four CNGCs under the respective native promoter. Thus, our findings reveal a type of ABA-activated PM Ca2+ channel comprising multiple CNGCs, which is essential for ABA-specific Ca2+ signaling of guard cells and ABA-induced stomatal closure in Arabidopsis.

Extracellular malate induces stomatal closure via direct activation of guard-cell anion channel SLAC1 and stimulation of Ca2+ signalling.

Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate-induced stomatal closure is not fully elucidated. We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate. A treatment with malate induced stomatal closure in Arabidopsis thaliana wild-type plants, but not in the mutants deficient in the slow (S-type) anion channel gene SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1). The treatment with malate increased S-type anion currents in guard-cell protoplasts of wild-type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S-type currents of constitutively active mutants of SLAC1, which have kinase-independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild-type Arabidopsis guard cells and the malate-induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator. These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure.

Malate induces stomatal closure via a receptor-like kinase GHR1- and reactive oxygen species-dependent pathway in Arabidopsis thaliana.

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells.

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening

Stomata regulate plant water use and photosynthesis by controlling leaf gas exchange. They do this by reversibly opening the pore formed by two adjacent guard cells, with the limits of this movement ultimately set by the mechanical properties of the guard cell walls and surrounding epidermis.1,2 A body of evidence demonstrates that the methylation status and cellular patterning of pectin wall polymers play a core role in setting the guard cell mechanical properties, with disruption of the system leading to poorer stomatal performance.3-6 Here we present genetic and biochemical data showing that wall arabinans modulate guard cell flexibility and can be used to engineer stomata with improved performance. Specifically, we show that a short-chain linear arabinan epitope associated with the presence of rhamnogalacturonan I in the guard cell wall is required for full opening of the stomatal pore. Manipulations leading to the novel accumulation of longer-chain arabinan epitopes in guard cell walls led to an increase in the maximal pore aperture. Using computational modeling combined with atomic force microscopy, we show that this phenotype reflected a decrease in wall matrix stiffness and, consequently, increased flexing of the guard cells under turgor pressure, generating larger, rounder stomatal pores. Our results provide theoretical and experimental support for the conclusion that arabinan side chains of pectin modulate guard cell wall stiffness, setting the limits for cell flexing and, consequently, pore aperture, gas exchange, and photosynthetic assimilation.

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors ofABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA‐induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (−)‐catechin gallate (CG) and (−)‐gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage‐dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA‐induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (I Ca) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co‐treatment with CG/GCG and ABA suppressed ABA‐induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark‐induced stomatal closure or light‐ and fusicoccin‐induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+‐related signaling pathways. This study further supports the crucial role of I Ca channels of the plasma membrane in ABA‐induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress‐induced signal transduction pathways.

Structure of the Arabidopsis guard cell anion channel SLAC1 suggests activation mechanism by phosphorylation.

Stomata play a critical role in the regulation of gas exchange and photosynthesis in plants. Stomatal closure participates in multiple stress responses, and is regulated by a complex network including abscisic acid (ABA) signaling and ion-flux-induced turgor changes. The slow-type anion channel SLAC1 has been identified to be a central controller of stomatal closure and phosphoactivated by several kinases. Here, we report the structure of SLAC1 in Arabidopsis thaliana (AtSLAC1) in an inactivated, closed state. The cytosolic amino (N)-terminus and carboxyl (C)-terminus of AtSLAC1 are partially resolved and form a plug-like structure which packs against the transmembrane domain (TMD). Breaking the interactions between the cytosolic plug and transmembrane domain triggers channel activation. An inhibition-release model is proposed for SLAC1 activation by phosphorylation that the cytosolic plug dissociates from the transmembrane domain upon phosphorylation, and induces conformational changes to open the pore. These findings facilitate our understanding of the regulation of SLAC1 activity and stomatal aperture in plants.

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues.

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.